Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

IgG antibodies against the 2,3-di-o-acyltrehalose glycolipid of Mycobacterium tuberculosis were determined in a set of 49 sera from patients with pulmonary tuberculosis and 65 negative control subjects. We compared a conventional ELISA method using a beta-galactosidase anti-human IgG conjugate developed with ONPG, with an amplification ELISA system constituted of an anti-human IgG biotinylated conjugate, a streptavidin-alkaline phosphatase complex, and NADP as a substrate. The resulting NAD was measured by using a redox enzymatic recycling system of alcohol dehydrogenase, diaphorase and iodonitrotetrazolium as chromogen. With specificity set at 92.31% in both methods, we obtained a sensitivity of 42.86% in the conventional method and a sensitivity of 61.22% in the amplified method. We conclude that by using a more sensitive method we can detect cases that otherwise could be identified as false negatives.
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PMID:Development of an enzyme-linked immunosorbent assay (ELISA) combined with a streptavidin-biotin and enzyme amplification method to detect anti-2,3-di-o-acyltrehalose (DAT) antibodies in patients with tuberculosis. 782 39

Rat liver microsomes and, to a lesser extent, nuclei were previously shown to produce reactive oxygen species at elevated rates after chronic ethanol treatment. The ability of intact rat liver mitochondria to interact with iron and either NADH or NADPH, and the effects of ethanol treatment, on production of reactive oxygen intermediates was determined. In the presence of ferric-ATP, NADH or NADPH catalyzed mitochondrial lipid peroxidation. Rates were elevated two- to threefold with mitochondria from ethanol-fed rats with both reductants. Mitochondrial lipid peroxidation was insensitive to superoxide dismutase, catalase, or hydroxyl radical scavengers but was sensitive to GSH and anti-oxidants such as trolox. Mitochondrial generation of hydroxyl radical-like species (assayed by oxidation of chemical scavengers) was increased after chronic ethanol treatment, as was H2O2 production. Modifiers of mitochondrial metabolism such as rotenone, cyanide, or an uncoupling agent, had no effect on mitochondrial production of reactive oxygen intermediates. The membrane-impermeable thiol reagent, p-chloromercuribenzoate, was complete inhibitory with both mitochondrial preparations. The activity of the rotenone-insensitive NADH-cytochrome c reductase, an enzyme of the outer mitochondrial membrane, was increased 40 to 60% by the ethanol treatment. These results suggest that NADH acting via the outer membrane NADH reductase can catalyze an iron-dependent production of oxygen radicals by rat liver mitochondria. The outer mitochondrial membrane fraction, prepared by digitonin fractionation, displayed increased rotenone-insensitive NADH-cytochrome c reductase activity after ethanol treatment and was more reactive in catalyzing scission of pBR322 DNA from the supercoiled form to the open circular forms. Rates of oxygen radical production by mitochondria and the extent of increase produced by chronic ethanol treatment are similar to those previously found with microsomes when NADH is the cofactor. Oxidation of ethanol by alcohol dehydrogenase generates NADH, and NADH-dependent production of reactive oxygen species by various organelles is increased after chronic ethanol treatment. These acute metabolic interactions coupled to induction by chronic ethanol treatment may play an important role in the development of a state of oxidative stress in the liver by ethanol.
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PMID:Increased production of reactive oxygen species by rat liver mitochondria after chronic ethanol treatment. 813 51

Aurintricarboxylic acid (ATA), an inhibitor of Ca(2+)-dependent endonuclease activity, is often used to implicate a role for increased intracellular calcium in mechanistic toxicology studies. We report here on the ability of ATA to inhibit the activity of several NAD(H)/NADP(H)-requiring enzymes (purified or cellular homogenates), including lactic dehydrogenase, alcohol dehydrogenase, cytochrome c reductase, ethoxycoumarin o-dealkylase, isocitric dehydrogenase, glutathione reductase and glucose-6-phosphate dehydrogenase. These results were compared with the ability of ATA to inhibit micrococcal nuclease and rat liver Ca(2+)-dependent endonuclease activity in similar incubations. With the exception of alcohol dehydrogenase, ATA was a potent inhibitor of each of the purified enzymes, with IC50s ranging from 0.5 to 82 microM. In cell homogenates, however, ATA was from 10 to 100-fold less potent at inhibiting these enzymes. When exogenous protein was added to purified enzyme incubations, the effect of ATA was similarly diminished. Our results demonstrate that ATA inhibits a wide range of NAD(H)/NADP(H)-requiring enzymes in in vitro incubations using purified enzymes, but that the inhibitory effects are markedly reduced in incubations which more closely resemble a cellular milieu.
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PMID:Inhibition of NAD(H)/NADP(H)--requiring enzymes by aurintricarboxylic acid. 855 68

Tetrazolium-dye-linked alcohol dehydrogenase (TD-ADH) of Amycolatopsis methanolica could be resolved into three protein components, which have been purified. Each of the components has the ability to reconstitute TD-ADH activity when combined with the other two. Component 1 is identical to the previously characterized methanol:N,N'-dimethyl-4-nitrosoaniline oxidoreductase (MNO), a decameric protein with 50-kDa subunits, each carrying a tightly bound NADPH. Component 2 is a high molecular mass (> 640 kDa) protein with subunits of 44 kDa and 72 kDa, and which possesses a low tetrazolium-dye-linked NADH dehydrogenase activity. The protein contains a yellow chromophore of unknown identity. Component 3 is a low molecular mass (15 kDa) protein containing a 5'-deazaflavin and at least one other low-molecular-mass compound with properties similar, but not identical, to those of nicotinamide coenzymes. The results suggest that alcohol oxidation by the TD-ADH complex is carried out by component 1 (MNO), after which transfer of the reducing equivalents (mediated by component 3) occurs to component 2, which (in vitro) is linked to the tetrazolium dye. Fractionation of A. methanolica extracts showed that most of the 5'-deazaflavin was present in component 3. Other gram-positive bacteria having a TD-ADH complex also produced 5'-deazaflavin. It is concluded that oxidation of primary aliphatic alcohols by A. methanolica, and probably also by other gram-positive bacteria containing MNO or TD-ADH, proceeds via TD-ADH. The likeliness of 5'-deazaflavin participation in this process is discussed.
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PMID:Tetrazolium-dye-linked alcohol dehydrogenase of the methylotrophic actinomycete Amycolatopsis methanolica is a three-component complex. 924 38

NDI1 is the unique gene encoding the internal mitochondrial NADH dehydrogenase of Saccharomyces cerevisiae. The enzyme catalyzes the transfer of electrons from intramitochondrial NADH to ubiquinone. Surprisingly, NDI1 is not essential for respiratory growth. Here we demonstrate that this is due to in vivo activity of an ethanol-acetaldehyde redox shuttle, which transfers the redox equivalents from the mitochondria to the cytosol. Cytosolic NADH can be oxidized by the external NADH dehydrogenases. Deletion of ADH3, encoding mitochondrial alcohol dehydrogenase, did not affect respiratory growth in aerobic, glucose-limited chemostat cultures. Also, an ndi1Delta mutant was capable of respiratory growth under these conditions. However, when both ADH3 and NDI1 were deleted, metabolism became respirofermentative, indicating that the ethanol-acetaldehyde shuttle is essential for respiratory growth of the ndi1 delta mutant. In anaerobic batch cultures, the maximum specific growth rate of the adh3 delta mutant (0.22 h(-1)) was substantially reduced compared to that of the wild-type strain (0.33 h(-1)). This is consistent with the hypothesis that the ethanol-acetaldehyde shuttle is also involved in maintenance of the mitochondrial redox balance under anaerobic conditions. Finally, it is shown that another mitochondrial alcohol dehydrogenase is active in the adh3 delta ndi1 delta mutant, contributing to residual redox-shuttle activity in this strain.
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PMID:The mitochondrial alcohol dehydrogenase Adh3p is involved in a redox shuttle in Saccharomyces cerevisiae. 1094 11

cADP-ribose (cADPR) is a novel cyclic nucleotide derived from NAD(+) that has now been established as a general Ca(2+) messenger in a wide variety of cells. Despite the obvious importance of monitoring its cellular levels under various physiological conditions, its measurement has been technically difficult and requires specialized reagents. In this study a widely applicable high-sensitivity assay for cADPR is described. ADP-ribosyl cyclase normally catalyses the synthesis of cADPR from NAD(+), but the reaction can be reversed in the presence of high concentrations of nicotinamide, producing NAD(+) from cADPR stoichiometrically. The resultant NAD(+) can then be coupled to a cycling assay involving alcohol dehydrogenase and diaphorase. Each time NAD(+) cycles through these coupled reactions, a molecule of highly fluorescent resorufin is generated. The reaction can be conducted for hours, resulting in more than a thousand-fold amplification of cADPR. Concentrations of cADPR in the nanomolar range can be measured routinely. The unique ability of ADP-ribosyl cyclase to catalyse the reverse reaction provides the required specificity. Using this assay, it is demonstrated that cADPR is present in all tissues tested and that the levels measured are directly comparable with those obtained using a radioimmunoassay. All the necessary reagents are widely available and the assay can be performed using a multiwell fluorescence plate reader, providing a high-throughput method for monitoring cADPR levels. This assay should be valuable in elucidating the messenger role of cADPR in cells.
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PMID:A novel cycling assay for cellular cADP-ribose with nanomolar sensitivity. 1177 10

Twelve enzymes from mature pollen grains of maize were separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The separation in the second dimension was both in the presence and absence of sodium dodecyl sulfate (SDS). Ten of the investigated enzymes lost activity after separation in the presence of SDS, but those of esterases and acid phosphatase could be recovered. On the other hand, 2-D electrophoresis without SDS is suitable for the analysis of maize pollen pectinesterase, malate dehydrogenase, glutamic-oxalacetic transaminase, diaphorase, superoxide dismutase, and phosphoglucose isomerase. 1-D PAGE and isoelectric focusing (IEF) are sufficient to analyze glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, shikimic dehydrogenase, and glutamate dehydrogenase. The possibility of applying 2-D electrophoresis for the analysis of enzymes from single stigma and stigma exudate is dicussed.
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PMID:Maize pollen enzymes after two-dimensional polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate. 1182 13

Nicotinic acid-adenine dinucleotide phosphate (NAADP) is a novel nucleotide derived from NADP that has now been shown to be active in releasing Ca(2+) from intracellular stores in a wide variety of cells ranging from plant to human. Despite the obvious importance of monitoring its cellular levels under various physiological conditions, no assay has been reported for NAADP to date. In the present study, a widely applicable assay for NAADP with high sensitivity is described. NAADP was first dephosphorylated to nicotinic acid-adenine dinucleotide by treatment with alkaline phosphatase. The conversion was shown to be stoichiometric. NMN-adenylyltransferase was then used to convert nicotinic acid-adenine dinucleotide into NAD in the presence of high concentrations of NMN. The resultant NAD was amplified by a cycling assay involving alcohol dehydrogenase and diaphorase. Each time NAD cycled through these coupled reactions, a molecule of highly fluorescent resorufin was generated. The reaction could be performed for hours, resulting in more than a 1000-fold amplification. Concentrations of NAADP over the 10-20 nM range could be routinely measured. This novel cycling assay was combined with an enzymic treatment to provide the necessary specificity for the assay. NAADP was found to be resistant to NADase and apyrase. Pretreatment of samples with a combination of the hydrolytic enzymes completely eliminated the interference from common nucleotides. The versatility of the cycling assay can also be extended to measure nicotinic acid, which is a substrate in the synthesis of NAADP catalysed by ADP-ribosyl cyclase, over the micromolar range. All the necessary reagents for the cycling assay are widely available and it can be performed using a multi-well fluorescence plate reader, providing a high-throughput method. This is the first assay reported for NAADP and nicotinic acid, which should be valuable in elucidating the messenger functions of NAADP.
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PMID:A novel cycling assay for nicotinic acid-adenine dinucleotide phosphate with nanomolar sensitivity. 1211 13

The blood alcohol level cycle (BALC) of the intragastric tube feeding model first described by Tsukamoto et al., has three separate essential mechanistic components. The first is the requirement for an intact functioning thyroid. The evidence for this is that propylthiouracil or severance of the pituitary stalk completely prevents the cycle. What happens instead of the cycle is that the blood alcohol level rises to a lethal level when ethanol is given continuously at a dose of 11 g/kg/day by stomach tube. When excess thyroid hormone is given orally it markedly attenuates the cycle because it interferes with the changes in the level of thyroid hormone during the cycle. The second component is norepinephrine. Catecholamines are markedly elevated at the peaks of the cycle. Both propranolol and phenoxybenzamine, which are beta- and alpha-blockers, prevent the cycle. Also, when catecholamines are fed in excess in the form of ephedrine, the cycle is eliminated. The third element essential to the cycle is the generation of NAD to support the oxidation of alcohol by alcohol dehydrogenase. When complex I (NADH dehydrogenase) of the mitochondrial electron transport chain is inhibited by feeding rotenone, the cycle is totally eliminated and blood alcohol levels remain constant at 200 mg/%. Thus NADH increases and NAD decreases at the peak of the cycle. Without the fluxuation of NAD, ADH activity cannot fluctuate during the cycle and the cycle is prevented. The significance of the BALC in the understanding of alcohol liver disease pathogenesis is that there's a marked difference in the gene expression and liver toxicity when the peaks and troughs of the cycle are compared. The expression of 1000+ genes is either two-fold up or down regulated as determined by microarray analysis. At the peaks there is increased liver pathology, especially inflammatory changes in the liver associated with an increase of iNOS expression. The genes responsive to hypoxia inducible factor 1alpha (HIF1alpha) regulation are increased including the expression of erythropoietin, adrenomedullin and adrenergic receptor alpha 1a and d. The expression of prolyl hydroxylase, which destabilizes HIF1alpha, increases when the BAL drops to low levels during the cycle. The level of oxygen, as measured on the surface of the liver, is decreased at the peaks, compared to control livers. The NADH/NAD ratio is markedly increased and ATP levels are markedly decreased at the BAL peaks. Also, endotoxin in the blood is very high at the peaks and very low at the troughs. When the blood alcohol levels fall during the cycle, there is an increase in ALT, suggesting that reoxygenation from the hypoxic state at the peaks causes an ischemic reperfusion injury-like lesion in the liver. At this time there is also an increase in expression of many important enzymes such as manganese SOD. Genes such as c-fos and CTGF are increased in expression. These contrasting findings at the peaks and troughs indicate that the blood alcohol levels, which fluctuate up and down, change the gene expression and the pathology of the liver.
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PMID:The pathogenesis and significance of the urinary alcohol cycle in rats fed ethanol intragastrically. 1634 1

Extracellular and intraneuronal formation of amyloid-beta (Abeta) deposits have been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). However, the precise mechanism of Abeta neurotoxicity is not completely understood. Previous studies suggest that binding of Abeta with a number of targets have deleterious effects on cellular functions. It has been shown that Abeta directly interacted with intracellular protein ERAB (endoplasmic reticulum amyloid beta-peptide-binding protein) also known as ABAD (Abeta-binding alcohol dehydrogenase) resulting in mitochondrial dysfunction and cell death. In the present study we have identified another mitochondrial enzyme, ND3 of the human complex I, that binds to Abeta1-42 by the screening of a human brain cDNA library expressed on M13 phage. Our results indicated a strong interaction between Abeta and a phage-displayed 25 amino acid long peptide TTNLPLMVMSSLLLIIILALSLAYE corresponding to C-terminal peptide domain of NADH dehydrogenase, subunit 3 (MTND3) encoded by mitochondrial DNA (mtDNA). This interaction may explain, in part, the inhibition of complex I activity in astrocytes and neurons in the presence of Abeta, described recently. To our knowledge, the present study is the first demonstration of interaction between Abeta and one of the subunits of the human complex I.
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PMID:Identification of amyloid-beta 1-42 binding protein fragments by screening of a human brain cDNA library. 1638 38


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